This is a competitive renewal for ROI CA79976 "Dendritic cell-based genetic immunotherapy for melanoma" in which we request support for years 05-08. Our accomplishments in the previous funding period include: I. defining the immunological events taking place in a murine melanoma model using dendritic cell (DC) engineered with a defined tumor antigen MAKT-1, 2. completing a phase l/II clinical trial in melanoma patients receiving MAKT-127.35 peptide pulsed DC and 3. opening a gane therapy trial using adenovims (AdV) MAKT-l-transduced DC. Based on this pr6gress, we propose to continue our translational studies of genetic immunotherapy of human melanoma centered around three specific aims. Aim I: Genetic Immunotherapy in a CDS-Deficient Environment. We have made the remarkable and original observations that CD8 or Class I knock out mice immonized with AdVMARTl-transduced DC have superior levels of protection to B16 nu_lanoma than wild type (wt) mice. Since wt mice depleted of CD8 cells are unable to generate protective immunity, CD8 KO mice have developed a compensatory mechanism fi'om generating robust tumor immunity to DC vaccination. We present preliminary evidence that this antitumor immunity is mediated by a collaboration between effector cells of the innate (NK-like) and adaptive (CD4) arms of the immune systems. We propose to characterize the underlying mechanism. Aim 2: The Biology of Class I and Class II-Restritted T Cell Responses in AdVMART1/DC Immunized Patients with Melanoma. This clinical trial, in which patients with stage IV MAKT-l-positive melanoma are immunized with AdVMAKT1/DC, provides a unique opportunity to define immunological events triggered by genetic immunization to a defined "sell" tumor antigen. Only two epitopes have been described for this small protein- HLA-A2.1-restricted MAKT-lzT.35 and HLA-DK4 restricted MAKT- 15t-73.Using ELISPOT and tetramer assays for these class I and II epitopes, we will quantitate, isolate and study MART-l-reactive CD8 and CIM T cell in immunized patients. We will also study the role of determinant spreading and cross-presentation in clinical response, the biology of DC used for vaccination and the possible participation of innate (NK) immunity in DC-based immunotherapy. Aim 3:CTLA4 Blockade in Clinical Dendritic Cell-Based Immunotherapy. DC-based immunotherapy generates occasional but dramatic clinical antitumor responses. We have closely studied one subject in whom the administration of MART-I/DC vaccines was followed by a CTLA4 blocking antibody. Immunological analysis suggests that the antitumor imm,ne response initiated by the DC vaccines was maintained by CTLA4 blockade. To test this hypothesis, we have designed a phase II randomized trial with the primary goal of detecting the effect of MART-127.3y'DC + CTLA4 blockade on the frequency of melanoma antigen- specific activated T cells using ELISPOT assays. This trial will provide insight in the autoregulat0ry mechanisms that govern the activity ofDC-based imm_unotherapy. In summary, we propose to continue our translational prosram in genetic immunotherapy with an emphasis on immune mechanism and clinical hypothesis-testing.